Molecular and cellular mediators of inflammation play a major role in the initiation and progression of stroke. They also participate in the induction of tolerance to ischemia by sublethal preconditioning stresses. We have investigated inflammatory mediators in these three facets of stroke. We have also implicated activation of immune and inflammatory processes in the harmful effects of antibody therapy in a recent clinical acute stroke trial. Inhibition of tumor necrosis factor-alpha (TNF-alpha) with TNF-binding protein reduces brain infarct volume in middle cerebral artery occlusion (MCAO) models in the rat and mouse. In addition, TNF-binding protein attenuates the progressive impairment of microvascular perfusion that occurs during the early hours of focal brain ischemia. These findings implicate TNF-alpha as a mediator of progressive brain damage during acute stroke. Lipopolysaccharide (LPS) pretreatment has been demonstrated to induce tolerance to focal brain ischemia in the MCAO model in the SHR. TNF binding protein blocks this tolerance implicating TNF-alpha as the mediator. This form of tolerance also reduces the degree of microcirculatory perfusion impairment in brain. Preconditioning with TNF by intracisternal injection of TNF induces tolerance to ischemia in the Balb/C mouse. In vitro models comprising cellular elements of brain have been established in order to examine the mechanisms involved in the observed in vivo tolerance to ischemia of the brain pretreated either by tumor necrosis factor-alpha (TNF-alpha) or by hypoxia/ischemia. TNF-alpha pretreatment of rat cortical astrocytes and brain microvascular endothelial cells (BMEC) renders these cells unresponsive to TNF activation 24 hours later in terms of up-regulation and expression of ICAM-1 message and protein. Ceramide is a lipid messenger involved in TNF signaling. Addition of C- 2 ceramide 30 min before or even 1 hour after TNF exposure resulted in inhibition of up-regulation of ICAM-1 at both protein and mRNA levels. HPLC determination of endogenous ceramide concentrations in TNF-activated cortical astrocytes and BMEC demonstrated two peaks of ceramide release: an early increase at 15-20 minutes, and a late increase at 18-21 hours after TNF addition that was associated with tolerance. Inhibition of ceramide synthase with Fumonisin B1 attenuated TNF-induced preconditioning of astrocytes in a dose-dependent manner indicating that late ceramide release is a mediator of TNF-induced tolerance. Primary cultures of neonatal cortical neurons were subjected to 60 minutes of hypoxia and reoxygenated for various times. Cell death was quantitated using the ethidium iodide fluorescence exclusion technique. Pretreatment of neuronal cultures with short hypoxia (15 minutes) 24 hours prior to 60 minutes of hypoxia, protected neurons against hypoxia (number of dead cells was 9.4% versus 35% in non-pretreated cultures). Pretreatment with TNF (50 ng/ml) or the downstream signaling molecule,ceramide (10 micromolar), also protected cortical neurons against 60 minutes of hypoxia. TNF-preconditioning can induce tolerance to subsequent TNF and hypoxia exposure in BMEC, astrocytes and cortical neurons. Ceramide appears to be involved in the intracellular signaling pathways leading to tolerance of brain cells to ischemia. In in vivo studies, cell-permeable ceramides (C2- and C8-ceramide) have been demonstrated in an adult rat model of focal brain ischemia to reduce infarct volumes. In hypoxic/ischemic insults in neonatal rats, C2-ceramide significantly reduced brain damage, augmented Bcl-2 and Bcl-xl levels, and reduced TUNEL-positive cells. In cortical astrocytes from 2-3 day old Sprague-Dawley rats, preconditioning with TNF-alpha or ceramide to produce tolerance does not inhibit I-kappaB proteolysis, nuclear translocation of NF-kappaB or binding of the p65 subunit of NFkB to its consensus site on DNA. It does, however, prevent p65 phosphorylation and consequently disrupts the association of the coactivator protein, p300/CBP, with that subunit. The result is that expression of proinflammatory genes such as ICAM-1 is inhibited, but expression of cytoprotective genes such as manganese superoxide dismutase continues unabated. In bedside to bench studies, mouse anti-rat ICAM-1 antibody induced an inflammatory state in preclinical models of ischemic stroke that included activation of complement (C3a desArginine), granulocytes (CD11b up-regulation), and endothelium (E- and P-selectin expression). Serial administration of the antibody sensitized rats to produce anti-mouse antibodies and augmented infarct size in a focal brain ischemia model. Similar responses to the mouse anti-human ICAM-1 monoclonal antibody, Enlimomab, may have contributed to the adverse outcome of the Enlimomab acute stroke trial.